Photocatalytic Pt(IV)‐Coordinated Carbon Dots for Precision Tumor Therapy

Abstract Rapid, efficient, and precise cancer therapy is highly desired. Here, this work reports solvothermally synthesized photoactivatable Pt(IV)‐coordinated carbon dots (Pt‐CDs) and their bovine serum albumin (BSA) complex (Pt‐CDs@BSA) as a novel orange light‐triggered anti‐tumor therapeutic agent. The homogeneously distributed Pt(IV) in the Pt‐CDs (Pt: 17.2 wt%) and their carbon cores with significant visible absorption exhibit excellent photocatalytic properties, which not only efficiently releases cytotoxic Pt(II) species but also promotes hydroxy radical generation from water under orange light. When triggered with a 589 nm laser, Pt‐CDs@BSA possesses the ultrastrong cancer cell killing capacities of intracellular Pt(II) species release, hydroxyl radical generation, and acidification, which induce powerful immunogenic cell death. Activation of Pt‐CDs@BSA by a single treatment with a 589 nm laser effectively eliminated the primary tumor and inhibited distant tumor growth and lung metastasis. This study thus presents a new concept for building photoactivatable Pt(IV)‐enriched nanodrug‐based CDs for precision cancer therapy.


Characterizations. Transmission electron microscopy (TEM) was performed on an FEI
Tecnai-G2-F20 transmission electron microscope (200 kV). An Inca X-Max instrument was used for perform the EDS and elemental mapping. The XPS analyses were conducted on an ESCALAB 250Xi photoelectron spectrometer using Mo as the exciting source (Thermo Fisher Scientific). A Shimadzu UV-2600 spectrophotometer was used for collected the UVvisible absorption spectra, and the emission spectra were acquired from a Horiba Jobin Yvon Fluorolog-3 spectrometer (Xenon lamp excitation). The photoluminescence quantum yield of all nanomaterials was collected at room temperature on an Edinburgh FS5 spectrophotometer.
The fluorescence imaging of cell was collected from an Olympus FV1000 confocal laser scanning microscope. The laser (689 nm) was generated from cnilaser MD-655NM-HS-2W- Intracellular pH regulation of Pt-CDs and Pt-CDs@BSA. 2′,7′-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM) was used as a measure of intracellular pH value. Intracellular pH was determined from the pH-dependent ratio of emission intensity (535 nm) when the dye is excited at 488 nm (pH-dependent) versus the emission intensity when excited at its isobestic point of 440 nm (non-pH dependent) using a microplate reader (Absorbance 96, Byonoy GmbH). As previously described, a standard curve was calibrated with BCECF-AM-loaded dye over multiple pH values, and the linear range is 6.0 to 8.0 pH. Cells were incubated with 1 μM BCECF-AM for 20 minutes at 37°C and then washed three times with PBS prior to pH measurements.

Extracellular pH regulation of Pt-CDs and Pt-CDs@BSA.
Using 4T1 cells grown in 35mm 2 dishes, extracellular pH was measured using a microprobe-based pH system (Micro pH Electrode, Thermo Fisher). The electrode was calibrated with pH standards and placed in the media of the 35-mm 2 dishes containing the cells. pH measurements were made using an Accumet Ab150 pH meter (Fisher Scientific). Technology, USA) and anti-GAPDH (ab8245, abcam, USA). After the secondary antibody incubation, the membrane was washed three times with TBST and exposed with ECL (Millipore, USA).

Synthesis of Pt-based Pt-CDs and BSA trapped with
Propidium Iodide (PI) exclusion assay. 4T1 cells were seeded in 12 wells plate. After treatment, both suspended and sticked cells were collected and washed twice with cold PBS.
Cells were resuspended with 300 μL cold PBS containing 5 μg/mL PI (Sigma, USA) and subjected to flow cytometry for cell viability detection. Twenty thousand cells were examined, and the data were analyzed with FlowJo vX.07 software. intravenously injected with Pt-CDs and Pt-CDs@BSA (at an equivalent Pt dose of 2.5 mg/kg).
After 1, 6, 12, and 24 h, mice were sacrificed immediately. Major organs (heart, liver, spleen, lung, and kidneys) and orthotopic tumors were harvested, and the weight of all organs was recorded. The Pt content of tissues and tumors were performed by ICP-MS. Histopathological evaluation. For histological analysis, the organs (heart, liver, spleen, lung, and kidney) were fixed in 10% formalin, then embedded in paraffin. Slices of these organs 8 from the mice were stained with Hematoxylin & Eosin (H&E) and Immunohistochemistry (IHC). The histological sections were imaged by an optical microscope.

Live Animals Imaging and Tissue Distribution of Pt-CDs and
Statistical analysis. Data are presented as mean ± S.D. Statistical significance of differences among groups were performed by Student's t-test. The differences between experimental groups and control groups were considered statistically significant for P value <0.05.
9 Fig. S1. TEM image of the Pt-CDs in large area.                  Error bars indicate standard deviations (n = 5).